1. Field of the Invention
The present invention relates to a novel cyclomaltodextrin transferase (EC. 2, 4, 1, 19; hereinafter referred to as CGTase) which preferentially produces beta-cyclodextrin (hereinafter referred to as .beta.-CD) from starch and a method for preparation thereof. The present invention particularly relates to a novel CGTase which is obtained by cultivation of Bacillus coagulans and which preferentially produces .beta.-CD from starch or partial hydrolysates thereof to form a carbohydrate containing .beta.-CD as main components, and a method for preparation thereof.
2. Description of the Prior Art
A CGTase is an enzyme catalyzing the following reactions: CD producing reactions producing cyclodextrins (hereinafter referred to as CDs) by acting on the .alpha.-1,4-glucopyranoside bond of an .alpha.-1,4-glucan such as starch, amylose, amylopectin and glycogen or partial hydrolysate thereof; coupling reactions cleaving CDs and transferring their glycosyl residues to several glycosyl accepters such as sucrose and maltooligosaccharides; and disproportionation reactions producing maltooligosaccharides with various molecular weights by intermolecular transferring reaction of various maltooligosaccharides. .alpha.-CD (cyclohexaamylose) consisting of six glucose units, .beta.-CD (cycloheptaamylose) consisting of seven glucose units and .GAMMA.-CD (cyclooctaamylose) consisting of eight glucose units are well-known as CDs but branched CDs branching at the 6th-position of glucose of CDs through .alpha.-1,6-glucopyranoside bond are also manufactured.
These CDs have a character to make inclusion complexes by incorporating various organic compounds (referred to as guest compounds) into cavities thereof due to their specific structure exhibiting hydrophobic nature. As a result, CDs advantageously improve properties of the guest compounds such as stability to heat, oxygen or ultraviolet rays and solubility in water and various solvents. Thus CDs are utilized not only in the field of foods but also in the pharmaceutic, cosmetic, pesticide and various other fields.
As mentioned above, CDs are obtained as a mixture of various CDs and maltooligosaccharides by the action of CGTase on an .alpha.-1,4-glucan such as starch or partial hydrolysate thereof as a mixture of various CDs and maltooligosaccharides but the product yield and yield ratio of the CDs varies slightly depending on the origin of the CGTase. Several microorganisms are known as sources of CGTases but microorganisms belonging to Bacillus known as particularly good producers of a CGTase. For example, the following microorganisms are known: B. macerans (Biochemistry, vol. 7, p 114, 1968 and Agric. Biol. Chem., vol. 38, p 387, 1974 and ibid, vol. 38, p 2413, 1974); B. circulans (Amylase Symposium, vol. 8. p 21, 1973); B. mecaterium (Agric. Biol. Chem., vol. 38, p 387, 1974 and ibid, vol. 38, p 2413, 1974); B. stearothermophilus (Japanese Patent No. 947335, J. Jap. Soc. Starch Sci., vol. 29, p 7, 1982); and alkaliphilic Bacillus sp. (Die Starke, vol. 27, p 410, 1975, Agric. Biol. Chem., vol. 40, p 935, 1976 and ibid, vol. 40, p 1785, 1976).
Aside from microorganisms belonging to Bacillus, Klebsiella pneumoniae (Arch. Microbiology, vol. 111, p 271, 1977, Carbohydr. Res., vol. 78, p 133, 1980 and ibid, vol. 78, p 147, 1980) is reported to produce a CGTase. The CGTases produced by various microorganisms are classified into .alpha.-CD and .beta.-CD-forming types on the basis of the initial reaction product produced from starch. B. macerans and K. pneumoniae produce .alpha.-CD-forming type and B. circulans, B. megaterium and alkaliphilic Bacillus sp. produce .beta.-CD-forming type. Further, the CDs content of a reaction product produced by a CGTase of B. stearotheromphilus from starch is as follows: .beta.-CD&gt;.alpha.-CD&gt;.GAMMA.-CD. However, this CGTase is properly .alpha.-CD type because the initial reaction product of this CGTase is .alpha.-CD (Japanese Patent No. 947335 and J. Jap. Soc. Starch Sci., vol. 29, p 13, 1982).
A CGTase of .GAMMA.-CD producing type was recently found but its .GAMMA.-CD productivity is quite low, making it inappropriate for practical use (Denpun Kagaku, vol. 33, p 137, 1986 and Japanese Patent Disclosure No. 61-274680).
Proper use of a CGTase according to purpose is advantageous since CGTases from different sources exhibit different action depending on pH and temperature, as well as different product yields and yield ratios of CDs from starch. However, a CGTase with a suitable optimum temperature and pH or better stability to temperature and pH is easier to use industrially. In general, it is preferred that the optimum temperature of an enzyme used for production of a starch sugar such as amylase be between 65.degree. and 70.degree. C. and that the optimum pH thereof be in the weak acid range of from 4.5 to 6.5. One reason for setting the optimum temperature at 65.degree. to 70.degree. C. is to prevent microorganism pollution of the reaction solution. Microorganism pollution makes it necessary to use large amounts of alkaline agents such as sodium hydroxide to compensate for pH drop of the reaction solution which results from the pollution and also requires purification by, for example, deionizing and decoloring, which makes the process expensive and difficult. Further, use of CGTases having higher optimum temperature and stable temperature is not economical because it requires more energy to maintain the temperature of the reaction vessel and inactivate the enzymes.
Moreover, when CGTases having an optimum pH in the alkaline range are used, an isomeric reaction occurring simultaneously with the enzyme reaction and colorization reduce the product yield and make the purification operation complicated. Therefore use of such a CGTase is not economical.
As described above, CGTases useful for industrial processes have to exhibit not only high yield of CDs from starch but also suitable optimum temperature, optimum pH and temperature stability for practical use.